Thirteen glucose transporters (GLUTs 1-12 & HMIT) catalyze equilibrative sugar transport in humans. The GLUTs are members of a wider family of Major Facilitator Superfamily (MFS) transporters that catalyze transport of a diverse array of molecules but share a common architecture of 12 membrane-spanning helices (TMs) with cytoplasmic N- and C-termini. The GLUTs display distinctive substrate specificities and sensitivity to inhibition by small molecules and a range of transport behaviors including uniport, symport, antiport and oligomerization-dependent cooperativity. This proposal describes our continuing efforts to understand GLUT function in health and disease by determining the molecular basis of GLUT-mediated substrate transport. To do this, we must resolve: 1) Determinants of substrate specificity and their locations within GLUT architecture; 2) How some transporters catalyze uniport while others catalyze symport or antiport; 3) Which transporter elements promote transporter oligomerization and cooperativity. The GLUTs are well-suited to such studies being amenable to biochemical, molecular and kinetic analysis. This proposal exploits the structural similarities of the GLUT family of proteins to answer these questions. Our studies of GLUT1/GLUT3 chimerae show that GLUT1 TM9 is essential for GLUT1 oligomerization. Specific Aim 1 tests the hypothesis that TM9 presents a dimerization surface to adjacent GLUT1 subunits by investigating the ability of wt-TM9 and TM9 dimerization surface mutants to promote chimerae oligomerization by using novel co-immunoprecipitation, chemical crosslinking and TOXCAT assays. We also ask whether other GLUTs form homo- and hetero- oligomers. Recognizing that GLUT5 is a cytochalasn B (CB) insensitive fructose transporter that cannot transport 2-deoxy-D-glucose (2DOG) and GLUT1 is a CB-inhibited 2DOG transporter that does not transport fructose, Specific Aim 2 tests the hypothesis that specific clusters of GLUT sequence form exo- and endofacial substrate binding sites. GLUT1/GLUT5 chimerae will be assayed for GLUT5-sequence dependent loss of CB- inhibtion, loss of 2DOG transport and gain of fructose transport. Reciprocal gain of function studies with GLUT5/GLUT1 chimerae will verify the results. A novel, scanning factor Xa proteolysis technique will also define GLUT exo- and endofacial ligand binding sites. GLUTs 1 and 3 show trans-acceleration - accelerated sugar uptake in cells preloaded with sugar. GLUTs 2 and 4 do not. Specific Aim 3 tests the hypothesis that specific clusters of GLUT sequence determine antiport (trans-acceleration) and symport functions by scanning GLUT1/GLUT4 or GLUT1/GLUT2 chimerae mutagenesis. Chimerae will be assayed for GLUT2/4-sequence dependent loss of trans-acceleration and reciprocal gain of function studies with GLUT4/GLUT1 chimerae used to verify the results. Scanning GLUT1/HMIT chimerae will be used to expose HMIT domains required for H+:myo-inositol symport. Our results will be mapped onto GLUT1 structure to provide new insights into GLUT structure and function and will have broad significance to the MFS proteins that mediate organismal energy homeostasis, solute equilibrium and drug delivery/resistance. PUBLIC HEALTH RELEVANCE: Glycopenia (tissue glucose shortage) can have genetic, endocrine and pharmacologic origins, results in seizures, focal neurologic deficits, coma and, if uncorrected, impairs development. This proposal continues our efforts to understand how the family of human glucose transport proteins allows organs and cells to absorb glucose and other sugars from the blood. The insights we gain from these studies will impact our understanding of the wider family of Major Facilitator Superfamily transport proteins. Our long-term goal is to translate these insights into practical intervention in clinical glycopenia.